The presence and enumeration of microorganisms in industrial samples have been traditionally determined by growing the microorganisms on the surface of agar in Petri dishes and counting the colonies. In the last two decades, other tests have been practiced for clinical and industrial specimens. These methods are based on culturing the specimens in liquid media and monitoring the metabolites generated during the growth of the micro-organisms. Several systems, such as the BACTOMETER® (bioMerieux, Hazelwood, Mo., USA), BACTRAC® device (Sy-Lab, Neupurkersdort, Austria), MALTHUS SYSTEMS® device (Lab M, Crawley, UK) and the RABIT® device (Bioscience International, Bethesda, Md., USA), are based on monitoring the electrical properties of the media measured via two metallic electrodes immersed in the growth media. The conductivity and capacitance of the electrode-media combination is measured by applying AC electrical current via the electrodes in the liquid media.
A novel and practical approach of culturing and monitoring microorganisms (bacteria, yeasts and molds) in test samples in the presence of interfering materials has been developed and successfully commercialized utilizing optical indicator substrates. One such product has been demonstrated by Turner, et al. (U.S. Pat. No. 4,945,060), Calandra, et al. (U.S. Pat. No. 5,094,955), Thorpe, et al. (U.S. Pat. No. 5,162,229), Di Guiseppi, et al. (U.S. Pat. No. 5,164,796), and Turner, et al. (U.S. Pat. No. 5,217,876). The basic principle of this device is to affix a disposable sensor to the interior surface of a transparent container that can monitor pH changes in the liquid media or the production of CO2 when the microorganisms grow and metabolize. The sensor comprises a solid composition or membrane with an indicator substrate immobilized on or within it. The sensor is placed flush against the inside surface of a container, such that the indicator substrate is visible from outside, and sealed to prevent the interfering compounds from getting between it and the container surface. In these embodiments the sensor is separated from the specimen and its growth media by a membrane or solid layer that permits the passage of gas molecules but prevents passage of ions. These devices are therefore characterized by two distinctive phases: (a) liquid phase that includes the growth media where the specimen is incubated and (b) solid phase in which the indicator substrate is embedded. In these devices, no growth media is present in the solid phase and no indicator substrate is present in the media. Practically, since sensors are based upon diffusion of CO2 gas (U.S. Pat. No. 5,217,876), they do require that the container is sealed during the incubation time so that the generated gas is pressurized through the sensor and cannot escape the container (U.S. Pat. No. 4,945,060). Consequently, these devices are limited to the determination of presence or absence of microorganisms in the tested samples which is adequate for clinical and sterility tests. Due to the solid nature of the sensor, the diffusion rate of the metabolites to the sensor is quite slow and may not be consistent for duplicate samples and, consequently, is inadequate for enumeration tests. The clear advantage of these devices is that they can be thermally sterilized (e.g., using autoclave at 121° C.) and, consequently, can be used for highly demanding sterility tests.
Another practical approach has been developed by Eden, et al. (U.S. Pat. No. 5,366,873), which is most suitable for food, dairy, and beverage samples. With this approach, the test container contains two distinct phases: (a) liquid phase, which is a mixture of growth media and indicator substrate, and (b) semi liquid phase, comprising a semi-liquid matrix, such as agar, and identical liquid compounds present in the liquid phase. Liquid molecules and ions can quickly diffuse between the two phases which are in equilibrium. The diffusion rate is higher in this device relative to the Turner device (U.S. Pat. No. 4,945,060), and its consistency makes it adequate for enumeration tests. The disadvantage of this device is that the semi-liquid phase disintegrates in higher temperatures and, therefore, the device cannot be thermally sterilized. Consequently, it cannot be used for clinical and sterility tests. Another disadvantage of this device is that the agar occasionally gets dislodged during transportation.